The mammalian target of rapamycin (mTOR) is a central regulator of cell growth that is activated by a large number of cancer-promoting mutations. mTOR exists in two multi-protein complexes: mTORC1, which activates protein synthesis, and mTORC2, which is upstream of Akt-dependent anti-apoptotic pathways. The mTORC1 inhibitor Rapamycin and its analogues are in clinical trials against a variety of malignancies. While mTOR inhibition offers much promise as a chemotherapeutic strategy, the differential roles of mTORC1- and mTORC2-dependent pathways in carcinogenesis have only begun to be explored. A role for mTOR-controlled pathways in non-melanoma skin cancer (NMSC) has been suggested in recent clinical trials showing that renal transplant patients administered Rapamycin as an immune suppressant suffer from significantly fewer NMSCs compared to patients taking Calcineurin inhibitors. The central hypothesis of the proposed work is that both mTORC1 and mTORC2 activities within keratinocyte stem cells are essential for the initiation and promotion of NMSC. We show that topical application of Rapamycin to mouse skin inhibits epidermal hyperproliferation in response to the tumor promoter TPA and sensitizes keratinocytes to apoptosis following UV irradiation. It is known that mTORC2 can also respond to Rapamycin treatment, either by activation or inhibition, and mTORC2 is required for the development of prostate cancers dependent on Akt hyperactivation. Aberrant Akt activation has also been demonstrated in mouse models of NMSC. The proposed experiments will use a CreLoxP approach to differentiate between mTORC1- and mTORC2-specific effects in NMSC by creating mouse lines with conditional deletion of Raptor to abrogate mTORC1 or Rictor to abrogate mTORC2 in the skin. A number of studies have indicated that stem cells within the hair follicle bulge give rise to NMSC. Thus, mice with a floxed raptor or rictor allele will be crossed with K5CreERT2 mice, allowing Raptor or Rictor deletion to occur in the follicular bulge upon topical Tamoxifen application. We will use these mice to address the hypothesis that both mTORC1 and mTORC2 activities are required to maintain keratinocyte stem cell homeostasis and to become oncogenically transformed. Aim 1 will examine the effect of Raptor or Rictor deletion on the development of skin tumors using the classical DMBA/TPA initiation-promotion model, and will analyze the stem cell apoptotic response to DMBA. In Aim 2, changes in the stem and progenitor cell compartments that occur with loss of Raptor or Rictor will be evaluated. To measure the response to a primary oncogenic event, isolated Raptor ? ?Cre or Rictor / -Cre keratinocytes will be transduced with v-Ha-ras. Differentiation changes and the ability to form papillomas on nude mice will be assessed. Aim 3 will perform transcriptional profiling of stem cell-enriched Raptor / -Cre and Rictor / -Cre keratinocytes, allowing insight into other potential targets in NMSC. If our hypothesis is confirmed, these studies will provide a strong rationale for development of mTORC1- and mTORC2-specific inhibitors in skin and other epithelial tumors. PUBLIC HEALTH RELEVANCE: Our research studies the role of mTOR-dependent pathways in development of non-melanoma skin cancer. Since skin cancers are the most common form of malignancies world-wide, identification of possible targets for prevention and therapy is highly relevant to public health. Understanding the role of the mTORC1 and mTORC2 protein complexes is also of great importance because the mTOR inhibitor Rapamycin has shown great promise in chemotherapy of a variety of human cancers.